Transcutaneous auricular vagus nerve stimulation (taVNS) is an emerging neuromodulation technology that has been reported to be beneficial in the treatment of diseases by several studies, but its exact mechanism of action is still unclear. It has been demonstrated that taVNS can influence interoceptive signals. Notably, the processing of interoceptive signals is directly related to many diseases, such as depression, anxiety, and insomnia. The insula and the medial prefrontal cortex (MPFC) communicate during the bottom-up transmission of taVNS-induced signals, and both play a role in interoceptive signal processing. By focusing on the insula and MPFC, our research pioneers detail the potential interactions between interoceptive signal processing and the neuromodulation effects of taVNS, providing novel insights into the neurobiological mechanisms of taVNS. Two functional connectivity (FC) analyses (region of interest-based and seed-based) were used in this study. We observed that negative connectivity between the insula and the MPFC was significantly weakened following taVNS, while there were no statistical changes in the sham group. Our findings elucidate potential mechanisms linking vagal activity with intrinsic FC among specific brain regions and networks. Specifically, our results indicate that taVNS may enhance the ability to flexibly balance interoceptive awareness and cognitive experiences by modulating the FC between the insula and MPFC. The modulation effects may impact body-brain interactions, suggesting the mechanism of taVNS in therapeutic applications.